For the past several decades, molecular hybridization has been an extremely useful tool for identifying and analyzing specific nucleic acid sequences in complex mixtures. The basic technique has undergone various changes that permit the simultaneous analysis of multiple sequences in a single assay (multiplexing) with increased speed and sensitivity of detection.
The sensitivity of detection must be sufficient to allow sequences present in the initial sample in single or low copy number to be reproducibly detected and distinguished from background noise. The signal-to-noise problem has been addressed in various ways, e.g., by amplifying the target nucleic acid sequences, by enhancing the signal generated by molecules associated with the hybridized sequences, and by reducing non-specific background binding.
Various sandwich hybridization techniques have been developed to detect specific sequences in nucleic acids. These include: a one-step sandwich hybridization assay in which two probes, one immobilized and one labeled, are bound to non-overlapping sequences in the target (U.S. Pat. No. 4,563,419); an amplification assay in which a primary probe contains non-overlapping sequences which are respectively complementary to the target DNA and to multiple signal-generating probes (U.S. Pat. No. 4,882,269); the formation of a triple helix between a single-stranded target sequence and two complementary oligonucleotide probes (U.S. Pat. No. 5,772,081); multihybrid formation using probes capable of binding to more than one nucleic acid target sequence (U.S. Pat. No. 4,894,325; U.S. Pat. No. 5,487,973); and the use of multimers capable of hybridizing directly or indirectly to a target nucleic acid sequence and to a second oligonucleotide sequence that is capable of binding multiple labeled oligonucleotides (U.S. Pat. No. 5,124,246).
The invention described herein is a method for achieving signal amplification that requires little manual handling, uses simplified signal enhancement reagents and is capable of being used with "microchip"-based genetic screening and diagnostic applications.